DNA Extraction

We used the same beadbeating protocol for all sample types in order to isolate the impact of kit chemistries (buffers and reagents) on our results. A total of six stool samples, six sputum samples, and six dust samples were included in this comparison (Figure 1). Four replicates of each sample were placed into individual Lysing Matrix E tubes (MP Biomedicals) in the following amounts prior to DNA extraction: 100 mg of stool, 500 μL of sputum, and 200 mg of vacuumed dust. Lysing matrix E tubes were chosen as they contain 1.4 ceramic spheres, 0.1 mm silica spheres and one 4 mm glass bead; this range of bead sizes allows efficient lysis of diverse human and environmental sample types. Each replicate was then processed using each of the four extraction methods. To each tube, 750 μL of lysis buffer was added as follows: Cetyl Trimethyl Ammonium Bromide (CTAB) for Methods 1 and 2 below, PowerBead Solution/RNase A Solution for Method 3 below, ZymoBIOMICS Lysis Solution for Method 4 below. Beadbeating occurred for a total of 6 cycles at 7.0 m/s with each cycle lasting 30 s followed by 90 s pause. Pauses were incorporated to avoid overheating the sample due to friction generated by beadbeating. Sample lysate was then used for downstream extraction according to each protocol.

The KingFisher Flex benchtop automated extraction instrument (Thermo Fisher Scientific, Waltham) was used for high-throughput DNA extraction for Methods 2–4 as described below. In comparisons of different automatic extraction instruments, the Kingfisher platform has previously been shown to have superior yield for sequencing applications (Marotz et al., 2017). In this approach, the lysate after beadbeating for each sample is placed in 96-well plates with magnetic beads used to transfer samples between plates for binding, washing, and elution steps. Our comparison methods focused on magnetic bead-based extraction protocols due to the need for a high-throughput extraction pipeline and prior literature suggesting that DNA yields are improved with magnetic bead-based protocols compared to spin column-based protocols (Moeller et al., 2014; Dunbar et al., 2018).

Microbial DNA extraction was performed using a modified cetyltrimethylammonium bromide–polyethylene glycol (CTAB) phenol:chloroform extraction protocol used in urban asthma studies focused on the built environment microbiome as previously described (Fujimura et al., 2014; Lai et al., 2018). Briefly, this protocol involves the addition of CTAB for sample lysis followed by a heating step at 65°C for 15 min, addition of phenol:chloroform:isoamyl alcohol (25:24:1), beadbeating, then transfer of the supernatant to heavy phase-lock gel tubes (5Prime). One volume of chloroform is then added to each sample, which is centrifuged briefly. Linear acrylamide is added to the supernatant followed by a 2-h incubation with PEG at room temperature, washing with ice-cold 70%, air drying, and resuspension in molecular-grade H₂O.

Microbial DNA extraction was performed based on Technical Manual #TM473 available on www.promega.com/protocols. Modifications were made to the protocol for use with the KingFisher Flex rather than the Maxwell^® RSC instrument with kit chemistries similar to the Maxwell RSC PureFood GMO and Authentication Kit. Briefly, CTAB is added to sample aliquoted in beadbeating tubes, heated to 95°C for 5 min followed by beadbeating, addition of proteinase K, incubation at 70°C for 10 min, centrifuged, then the sample lysate transferred to 96-well plates for subsequent binding, washing, and elution steps on the KingFisher Flex. The kit binding buffer was replaced with 100% isopropanol in this protocol.

Microbial DNA extraction was performed using the Earth Microbiome Protocol (Marotz et al., 2017) with a modification to the beadbeating step as described above. A step-by-step description of this protocol is outlined in https://www.protocols.io/view/earth-microbiome-project-emp-high-throughput-htp-d-pdmdi46. Briefly, PowerBead Solution, RNase A, and SL Solution (a lysis butter) is added to each beadbeating tube followed by a heating step at 65°C for 10 min, beadbeating, transfer of the supernatant to 96-well plates for subsequent binding, washing, and elution steps on the KingFisher Flex.

Microbial DNA extraction was performed according to manufacturer instructions, with the exception of the beadbeating step as described above. This kit has similar chemistries to other ZymoBIOMICS kits including the Miniprep kit, Microprep kit, and 96 DNA kit, with an adaptation made for automated magnetic bead platforms. Briefly, lysis solution and proteinase K was added to each sample in beadbeating tubes, the sample was incubated at 55°C for 30 min, followed by beadbeating, transfer of the sample lysate to 96-well plates for subsequent binding, washing, and elution steps on the KingFisher Flex.